Transport across the plasma membrane is a critical feature of all cellular physiology. Many gatekeepers at the plasma membrane are assisted by ionic and electric gradients across the membrane. Cells expend enormous energy-- up to 50% of intracellular ATP-- for maintenance of such electrochemical gradients. Energy economics of charging the plasma membrane with an electrochemical gradient for such transport remains virtually unknown in malaria parasites. The fact that erythrocytic stages of malaria parasites derive their ATP mainly through substrate-level phosphorylation, eking out a mere two ATP molecules per glucose molecule, must place significant constraints on parasite energy utilization. This project seeks to explore an alternate and/or adjunct energy source for malaria parasites. Recent evidence shows that Plasmodium species contains two members (PfVP1 and PfVP2) of the plant-like energy- conserving, membrane-associated H*-pumping pyrophosphatases. The vacuolar pyrophosphatases (V-PPases) of plants couple the energy generated by hydrolysis of the phosphoanhydride bond of inorganic pyrophosphate (PP_) to pump H [unreadable] across the vacuolar membrane. In malaria parasites, preliminary data suggest that the enzyme is located within the parasite plasma membrane. This location would suggest that H[unreadable] translocation across the parasite plasma membrane could be energized through PP_ hydrolysis by the PPases in concert with ATP hydrolysis by the V-type ATPase. Because animal cells do not possess homologues of V-PPases, the presence of these enzymes in malaria parasites offers candidates for devising selectively toxic inhibitors. This project will undertake basic investigations on the biochemistry and cell biology of PfVP1 and PfVP2. Gene disruption approaches will be undertaken to assess contributions made by these molecules to the parasite physiology. The possibility that V-ATPase of malaria parasites may function work in reverse to synthesize ATP by using the proton motive force generated by the V-PPases under high energy demand will be explored. Furthermore, an unusual subunit configuration observed for the FoF1-ATP synthase of malaria parasites will be investigated to assess the contribution of this usually mitochondrial proton pumping complex to parasite physiology. Results from this project have a potential to require a major revision of our view of malaria parasite bioenergetics. Unique features of proton homeostasis and bioenergetics in malaria parasites likely to be uncovered in this project could form the basis for devising novel approaches to malaria control. PERFORMANCESITE(S) (organization, city, state) Department of Microbiology and Immunology Drexel University College of Medicine Philadelphia, PA 19129 KEY PERSONNEL. See instructions.Use continuationpagesas neededto provide therequiredinformationin the formatshown below. StartwithPrincipalInvestigator. List allotherkey personnelinalphabeticaol rder,last namefirst. Name Organization Role on Project Akhit B. Vaidya, Ph.D. MCP Hahnemann University Principal Investigator Lawrence W. Bergman, Ph.D. MCP Hahnemann University Co-Investigator Michael W. Mather, Ph.D. MCP Hahnemann University Co-Investigator DisclosurePermission Statement. Applicable to SBIPJSTTROnly. See instructions. [] Yes [] No [unreadable] PHS 398 (Rev. 05/01) Page _2 Form Page 2 [unreadable] [unreadable] Principal Investigator/Program Director (Last, first, middle): Vaidya, Akhil B. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description,